Tandem triad systems based on FRET for two-photon induced release of glutamate

Article abstract of DOI:10.1039/c3cc45812a

Tandem systems allowing enhanced two-photon (2P) absorption in a wavelength range permitting coupling of the primary excitation by energy transfer to an intramolecular cage known to have fragmentation properties suited to photolysis in neuroscience is demonstrated to lead to a 10-fold improvement in the 2P photolysis cross-section at experimentally compatible wavelengths. The Royal Society of Chemistry 2013.

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Tandem triad systems based on FRET for two-photon induced release of
glutamate
a
a
a
a
b
Sébastien Picard, Eduardo Jose Cueto-Diaz, Emilie Genin, Guillaume Clermont, Francine Acher,
c
a
David Ogden,* Mireille Blanchard-Desce*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Tandem systems allowing enhanced twoꢀphoton (2P) absorption in a wavelength range permitting
coupling of the primary excitation by energy transfer to an intramolecular cage known to have
fragmentation properties suited to photolysis in neuroscience is demonstrated to lead to a 10ꢀfold
improvement in the 2P photolysis crossꢀsection at experimentally compatible wavelengths.
1
0
1
b, 1c, 6
Caged compounds, which can be irreversibly triggered by light,
have attracted lot of interest over the years due to the many
charge redistribution responsible for large 2PA response.
Instead of trying to achieve both enhanced 2PA and efficient
applications they offer in the fast developing field of 50 uncaging within a single chromophoric system, we implemented
1
neurosciences. In particular, uncaging of the excitatory
neurotransmitter ꢀglutamate (Glu) has been widely used in the
an alternative approach where the two processes are performed by
two distinct units crafted to act in tandem.
1
2
2
3
3
4
4
5
0
5
0
5
0
5
L
activation and functional mapping of synaptic receptors and their
signalling pathways, in studies of dendritic integration and of
neuronal circuits. In parallel, twoꢀphoton (2P) excitation in the
redꢀNIR region has emerged as a major tool for neurosciences,
allowing much better spatial resolution deep within neural tissue
1
a, 1c
than is possible with 1P excitation.
Among the caging
systems applied in neuroscience, the 7ꢀnitroindolinyl (NI) and 4ꢀ
methoxyꢀ7ꢀnitroindolinyl (MNI) photoremovable carbonyl
protecting groups have become the most widely used because of
their resistance to hydrolysis, their fast (subꢀꢁs) release kinetics
Fig. 1 Design of synergic triad structures 2P uncaging of glutamate
2
and lack of pharmacological interference in neural systems.
However, although efficient in 1P photolysis, the 2P uncaging
crossꢀsection (δ = σ Φ , the product of the 2P absorption crossꢀ
u
2
u
section (σ ) and uncaging quantum yield (Φ )) remains relatively
2
u
low (δu = 0.06 GM at 720 nm) preventing efficient 2P photolysis
at nonꢀtoxic intensities and requiring high cage concentrations for
3
in vivo studies. Attempts to increase δu of NI derivatives via
structural variations in the NI chromophore gave only small
4
improvements of the 2P uncaging efficiency. An approach in
which the triplet states mediating NI photolysis were populated
from an intramolecular sensitizer was found to be efficient but
blueꢀshifted the wavelength range for 2P photolysis beyond that
5
available from commonly used Tiꢀsapphire lasers. Thus there is
a strong motivation for the development of novel twoꢀphoton
sensitive caged systems combining large δu in the redꢀNIR region
with fast photolysis reactions for timeꢀresolved release of
neurotransmitters, and with excellent dark stability in biological
conditions.
Most 2P uncaging systems developed so far relied on common
uncaging systems (such as nitrobenzylꢀ or coumarinylmethylꢀ
based cages) by including them in dipolar or quadrupolar
conjugated compounds where they take part in the intramolecular
5
6
5
0
Scheme 1 Reagents and conditions: i) PdCl
2
(Ph
3
P)
2
, CuI, CsF, THF/Et
3
N,
rt, 3a 46%, 3b 70%; ii) NaH, propargyl bromide, DMF, rt, 4a 87%, 4b
7
9
1%; iii) NaBH
5% (over 2 steps); v) Cu(NO
, DMSO, 110 °C, then vii) KOH, THF/MeOH/Water,
rt, 45% (over 2 steps); viii) oxalyl chloride, DMF, THF, rt, 57%; ix)
sodium ꢀascorbate, CuSO , DMSO, rt, 10a 72%, 10b 80%.
3
CN, AcOH, rt, then iv) DMAP, Et
3 2 2 2
N, Ac O, CH Cl
, rt,
3
)
2
, CH Cl /Ac O, rt, 75% (5 and 7ꢀnitro,
2
2
2
ratio 1:1); vi) NaN
3
L
4
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Table 1 . Photophysical characteristics of the chromophores 3aꢀb and the synergic triad systems 10a-b in acetonitrile
max
abs
max
max
max
TPA
max
λ
Log(ε
)
λ
εm
Stokes shift
[10 cm ]
b)c)
λ
σ
2
at λ2PA
[GM]
48
83
42
ꢀ
1
ꢀ1
3
ꢀ1 a)
Φ
f
[
nm]
[M .cm ]
[nm]
383
411
382
410
[nm]
714
721
710
720
3
3
a
b
354
358
355
358
4.81
4.88
4.93
4.90
2.1
3.6
2.0
3.5
0.67
0.59
0.10
0.07
1
1
0a
0b
76
a
b
c
Stokes shift = 1/λabs – 1/λem ; Φ
f
= fluorescence quantum yields ; standard : quinine (Φ=0.546) in 0.5M aq H
2
SO
4
The quadrupolar module is designed for improved 2PA in the
wavelength range <800 nm, so as not to interfere in the range
used for 2P imaging. We chose to focus on the 4ꢀalkyloxyꢀ
spectrum of the NI chromophore, favouring FRET from the 2PA
module to the nearby NI.
5
0
5
0
5
0
5
0
5
0
7
substituted NI uncaging unit similar to MNIꢀGlu in order to retain
its hydrolytic stability and fast uncaging kinetics, of crucial
importance for neuroscience investigations. Here we report the
first synergic systems based on this strategy. They are composed
1
.0
0.8
.6
3
3b
a
NI acetate
1
1
2
2
3
3
4
4
5
0
7
of a hydrophilic quadrupolar platform built from a fluorenyl core
covalently grafted to two NIꢀcaged glutamate moieties (Fig 1).
We chose to use ethynyl linkers in order to preserve the
0.4
0.2
0.0
7
a
photostability of the 2PA chromophoric module while adjusting
its emission properties to allow FRET to the nitroindolines. Such
synergic systems can in principle release two Glu
neurotransmitters per molecule upon irradiation.
300
350
400
450
500
550
600
5
5
λ (nm)
Fig. 2 Normalised absorption and fluorescence spectra of chromophoric
moieties 3a, 3b and NI in acetonitrile.
The synthesis of targeted synergic structures 10a and 10b is
detailed in Scheme 1. The quadrupolar chromophoric modules
bearing either two electronꢀreleasing (D) or electronꢀwithdrawing
8
0
0
(A) groups, 4a and 4b, were prepared in two steps by means of an
3a
3
b
in situ deprotection / double Sonogashira coupling of the known
6
8
2
,7ꢀdiiodofluorene derivative 2 with the corresponding silylated
alkynes 1a or 1b followed by the propargylation of the terminal
hydroxyl groups in the presence of propargyl bromide with NaH
in DMF. In parallel, the 4ꢀsubstituted NIꢀGlu subunit 9 was
40
20
synthesised following a sixꢀstep sequence from the known 4ꢀ(3ꢀ
0
9
chloropropoxy)indole 5 which was first reduced with NaBH CN
3
720
740
760
λ (nm)
780
800
to afford, after acetylation, the protected indoline 6 with 95% of
yield. A nitration reaction in the presence of copper nitrate was
then applied to convert the intermediate 6 into an unseparable
Fig. 3 2PA spectra of chromophoric moieties 3a and 3b in acetonitrile.
10
60 As shown in Table 1, compounds 10a and 10b show markedly
reduced fluorescence compared to their quadrupolar modules 3a
and 3b, respectively. This indicates that competing processes
occur after excitation of the quadrupolar module. Since
electrochemical studies indicate that intramolecular electron
transfer is not favoured, we derived (majored) FRET quantum
efficiencies values of 85% and 88%, for compounds 10a and 10b,
using equation (1):
mixture of 5 and 7ꢀnitro product (ratio 1:1) with 75% of yield.
Substitution of the chlorine atom by an azido moiety followed by
basedꢀpromoted deprotection of the acetyl group afforded the
desired derivative 7 as a pure regioisomer. The NIꢀGlu 9 was
further obtained by coupling compound 7 and the acyl chloride
generated in situ from the corresponding protected glutamate 8.
The MNIꢀGlu triads 10a and 10b were finally synthesised in very
good yields using a twoꢀfold Huisgen 1,3ꢀdipolar cycloaddition
of azide 9 with bisꢀalkyne 4.
6
7
7
8
5
0
5
0
ΦΕΤ = 1ꢀ(
'D and ΦD are the donor (quadrupoles) fluorescence
quantum yields in the absence and presence of the acceptor (NI),
Φ
'D/ ΦD
)
(1)
where
Φ
The photophysical characterisation and 2PA properties of the
chromophoric quadrupolar modules 3a-b and the related synergic
systems 10a-b are shown in Table 1 and Figures 2 and 3. All
compounds display an intense absorption band in the near UVꢀ
region with maxima located around 355 nm and high molar
11
respectively.
In addition chromophores 3a and 3b were found to show
markedly enhanced 2P absorption responses in the NIR region
max
4
ꢀ1
ꢀ1
with respective
σ
2
values of 48 and 83 GM at 720 nm (Figure
extinction coefficients ranging from 6.4 to 8×10 M cm , at least
one order of magnitude larger than the MNI cages (peak at 340
3) which are 70ꢀfold to 118ꢀfold larger than that of MNI (0.7
GM). Hence 2PA modules 3 in tandem compounds 10 possess
the required features to behave as suitable twoꢀphoton absorbers
below 750 nm (as confirmed by their 2PA response, Table1)
while permitting FRET to NI uncaging modules. Firstly,
comparative 2P photolysis experiments were conducted on triad
3
ꢀ1
ꢀ1
nm 4.3×10 M cm ). Quadrupolar chromophores 3a and 3b
exhibit also intense fluorescence emission in the near UVꢀblue
visible region with fluorescence quantum yields of 0.67 and 0.59,
respectively. Although small, an overlap is observed between the
emission spectra of fluorophores 3a or 3b and the absorption
2
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DOI: 10.1039/C3CC45812A
systems 10a, 10b and NI reference sample 9, using a Tiꢀsapphire
We thank G. Garivet, V. Hugues, E. Esposito and D. Rigault for
laser at 740 nm (Figure 4). 2P uncaging was monitored by reverse 50 assistance in synthesis, 2P excitation experiments, 2P uncaging
phase HPLCꢀMS to quantify the extent of reaction.
measurements and HPLC.
m
V
bar
B.Conc
Detector
Detector
A
A
Ch1 254nm
Ch2 365nm
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
1000
950
900
850
800
750
700
650
600
550
Notes and references
a
naproxen
Univ. Bordeaux, Institut des Sciences Moléculaires, CNRS UMR 5255,
3
51 Cours de la Libération, F-33405 Talence Cedex, France. Fax: (+33)
5
5
5-40-00-69-94; E-mail: m.blanchard-desce@ism.u-bordeaux1.fr
b
CNRS UMR8601, Université Paris Descartes, Sorbonne Paris Cité, 45
500
450
400
rue des Saints-Pères, 75270 Paris cedex 06, France
c
FmocꢀGlu(OH)ꢀOFm
350
CNRS UMR8118, Université Paris Descartes, Sorbonne Paris Cité, 45
300
250
200
150
100
50
0
rue des Saints-Pères, 75270 Paris cedex 06, France. E-Mail:
60 David.Ogden@Parisdescartes.fr
† Electronic Supplementary Information (ESI) available: [Full
experimental procedures and analysis]. See DOI: 10.1039/b000000x/
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P. Klán, T. Šolomek, C. G. Bochet, A. Blanc, R. Givens, M. Rubina,
-
5
-
10
15
-
-
min
50
3.5
4.0
4.5
5.0
5.5
6.0
6.5
5
Fig. 4 Results obtained from the twoꢀphoton photolysis of compounds 9,
0a and 10b after 24 hours of irradiation at 740 nm (left) HPLC
chromatogramm obtained for the synergic system 10b; (right) plot of the
relative amount of released glutamate in the three cases (arbitrary units
are used).
1
65
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V. Popik, A. Kostikov and J. Wirz, Chem. Rev., 2013, 113, 119ꢀ191
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1
1
2
2
3
3
4
4
0
5
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5
The production of the photoꢀreleased glutamic acid bearing the
UVꢀresponsive fluorenyl moieties was monitored by UV
absorption (detection at 254 nm). We chose to use naproxen as an
internal reference to allow the determination of the concentration
of uncaged glutamate during the time course of the reaction.
Whereas almost no fragmentation was recorded after 24 hours of
2
001, 112, 29ꢀ42; e) J. Morrison, P. Wan, J. E. T. Corrie and G.
7
8
8
9
9
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0
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P irradiation of the NI reference sample 9, a noticeable HPLC
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peak corresponding to the photoꢀreleased glutamic acid was
observed in both synergic samples 10a and 10b. Very
interestingly, comparison of the percentage of released glutamate
in the different experiments showed that glutamate uncaging
efficiencies of the synergic systems 10a and 10b are respectively
3
4
.
.
N. Kiskin, R. Chillingworth, J. McCray, D. Piston and D. Ogden,
Eur. Biophys. J., 2002, 30, 588ꢀ604.
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Davies, M. Matsuzaki, M. Paukert, H. Kasai and D. E. Bergles, J.
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1
1ꢀfold and 15ꢀfold greater compared to the NI cage 9.
Compound 10b which has the larger 2PA response also lead to
the best 2P uncaging efficiency. Importantly, the synergic caged
MNIꢀGlu compounds 10a-b displayed no dark hydrolysis under
these conditions. In order to get an evaluation of the twoꢀphoton
sensitivities, the absolute measurement of δu of compound 10b
was further performed using a Tiꢀsapphire laser with excitation at
5. a) G. Papageorgiou, M. Lukeman, P. Wan and J. E. T. Corrie,
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7
30 nm (see SI). This led to (minored) δu value of about 0.5 GM
at 730 nm. This is consistent with an enhancement of about one
order of magnitude compared to MNIꢀGlu in the same conditions.
In conclusion, the triad systems investigated here, which allow
a oneꢀorder of magnitude enhancement in 2P uncaging sensitivity
compared to MNI provide proof of concept that the FRET route
is highly promising for the design of effective 2P cages below
00 nm, allowing combined 2P bioimaging at λ2PE>800 nm. This
novel route will be further generalised to other caging groups and
caged bioactive molecules.
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This work was supported by a funding from the European
Community’s Seventh Framework Programme under TOPBIO
projectꢀgrant agreement n. 264362. We also acknowledge
financial support from Agence Nationale pour la Recherche 110
Grant 2010 ANRꢀ10ꢀBLANꢀ1436), the Federation du Recherche
(
1
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This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3